The development of an effective vaccine is the most plausible approach to eliminate the HIV/AIDS pandemic. The detection of broadly neutralizing antibody (bNABs) responses is critical for vaccine development. In this grant application, we propose to develop a novel sensitive method for bNAB detection and quantification ? An HIV Rev-dependent, A3R5-derived, reporter cell line, Rev-A3R5 (NanoLuc/HaloTag). A3R5 is a T lymphoblast that expresses physiologically relevant levels of the HIV receptors, CD4 and CXCR4/CCR5, and is highly susceptible to X4 and R5 viruses. A3R5 has also been shown to be the optimal cell line currently available for bNAB detection. We plan to engineer A3R5 to carry two highly sensitive reporters: NanoLuc, the most sensitive luciferase currently available, and HaloTag, the most flexible fluorescent reporter. Both reporters are engineered to exclusively depend on HIV and Rev for expression, eliminating non-specific noises from environmental stimuli. This novel system will provide unmatched specificity and sensitivity, providing a new standard for NABs detection and quantification. We plan to carry out two specific aims in the phase I stage. Aim 1 will be to create a high-sensitivity, A3R5-derived, Rev-dependent reporter cell line, Rev- A3R5, for bNAB detection. We will first construct the Rev-dependent vector, pNL-NanoLuc-HaloTag- RRE, for assembly into lentiviral viral particles, which will be used to transduce A3R5 cells. Subclones will be selected for optimal performance in NAB detection. Aim 2 will be to characterize and validate Rev-A3R5. We will test the specificity of the reporter cells. Additionally, Rev-A3R5's susceptibility to primary viral isolates will be tested. We will also perform bNAB screen using a panel of antibodies and patient sera to optimize the system for Phase II development. We aim to develop the system into a standard assay product for academic and industry laboratories for bNAB screening and validation.